Treatment for attention-deficit hyperactivity disorder

ABSTRACT

A method for treating Attention Deficit/Hyperactivity Disorder (ADHD) in humans and the symptoms associated therewith, inattentiveness, and hyperactivity with impulsivity, using eltoprazine and related compounds is provided.

FIELD OF THE INVENTION

The present invention is directed to a novel method of treatingAttention-Deficit/Hyperactivity Disorder (“ADHD”). This invention alsorelates to improving cognitive functioning.

BACKGROUND OF THE INVENTION

Attention-Deficit/Hyperactivity Disorder (ADHD) is a behavior disordercharacterized by problems with control of attention andhyperactivity-impulsivity. The attentional difficulties and impulsivityassociated with ADHD have been persuasively documented in laboratoryinvestigations using cognitive tasks. Although these problems typicallypresent together, one may be present without the other to qualify for adiagnosis (Am. Psychiatric Assoc. Diagnostic and Statistical Manual ofMental Disorders, 4^(th) Ed., Text Revision, 2000) (DSM-IV-TR).Generally, attention deficit or inattention becomes apparent when achild enters elementary school. A modified form of the disorder canpersist into adulthood (Am. Psychiatric Assoc. Diagnostic andStatistical Manual of Mental Disorders, 3^(rd) Ed., 1987). With respectto the attention component, the child is easily distracted by outsidestimuli, neglects finishing tasks, and has difficulty maintainingattention. Regarding the activity component, the child is often fidgety,impulsive, and overactive. The symptoms of ADHD may be apparent as youngas preschoolers and are virtually always present prior to the age of 7(Halperin et al., J. Am. Acad. Child Adolescent Psychiatry,32:1038-1043, 1993).

According to the DSM-IV-TR, diagnostic criteria forAttention-Deficit/Hyperactivity Disorder relate to symptoms associatedwith inattention and/or hyperactivity-impulsivity. Three subtypes ofADHD are diagnosed based on the predominant symptoms presented.

Many of the symptoms that are characteristic of ADHD occur occasionallyin normal children. Children with ADHD, however, exhibit these symptomsfrequently, which tends to interfere with the child's day to dayfunctioning. Such children are often challenged by academicunderachievement because of excitability and impaired interpersonalrelationships.

ADHD affects 2-6% of grade school children. Pediatricians report thatapproximately 4% of their patients have ADHD; however, in practice thediagnosis is made in children who meet several, but not all of thediagnostic criteria that is recommended in DMS-IV-TR (Wolraich et al.,Pediatrics, 86(1):95-101, 1990). Boys are four times more likely to havethe disorder than girls and the disorder is found in all cultures (Ross& Ross, Hyperactivity, New York, 1982).

Psychomotor stimulants are the most common treatment for ADHD. Safer &Krager (1988) reported that 99% of the children with ADHD were treatedwith stimulants, of which 93% were given methylphenidate hydrochloride(Ritalin), and the remainder were given dextroamphetamine sulfate(d-amphetamine) or pemoline (Safer & Krager, J.A.M.A., 260:2256-2258,1988). Four separate psychostimulant medications consistently reduce thecentral features of ADHD, particularly the symptoms of inattention andADHD associated hyperactivity-impulsivity: methylphenidate,d-amphetamine, pemoline, and a mixture of amphetamine salts (Spender etal., Arch. Gen. Psychiatry, 52:434-443, 1995). These drugs block uptakesites for catecholamines on presynaptic neurons or stimulate the releaseof granular stores of catecholamines. They are metabolized and leave thebody fairly rapidly, and have a therapeutic duration of action of 1 to 4hours. The psychostimulants do not appear, however, to make long-termchanges in social or academic skills (Pelham et al., J. Clin. ChildPsychology, 27:190-205, 1998). Stimulants are generally started at a lowdose and adjusted weekly. Common stimulant side effects includeinsomnia, decreased appetite, stomachaches, headaches, and jitteriness.Psychostimulants also have the potential for abuse, because they areaddictive. Thus, current methods of treating ADHD provide inadequatetreatment for some patients and/or have side effects that limit theirusefulness.

Children who cannot tolerate psychostimulants often use theantidepressant bupropion. While bupropion is not as effective asstimulants, it may be used as an adjunct to augment stimulant treatment.

Castellanos et al. concluded that ADHD is a genetically programmeddisorder of brain development resulting from altered function of thefrontal-striatal-pallidal-thalamocortical loops which regulate cognitiveprocesses, attention, and motor output behaviors (Castellanos et al.,Arch. Gen. Psychiatry, 53: 607-616, 1996). Although the precise etiologyof ADHD is unknown, neurotransmitter deficits, genetics, and perinatalcomplications have been implicated.

Individuals with ADHD have been reported to have impairments in theirability to perceive intervals of time (Conners & Levin, Psychopharmacol.Bulletin, 32(1):67-73, 1996). Time perception is a useful measure ofcognitive function, sensitive to dopaminergic and cholinergicmanipulations in animals and humans. As in all behavioral tasks, severalprocesses underlie good steady state performance in a temporal task.These behavioral tasks include: attention, motivation, short and longterm memory, motor coordination, and instrumental learning. Scaling,discrimination, and reproduction are the three main types of temporaltasks that have been identified. In scaling, subjects must, for example,categorize a stimulus into a given set of categories (“that was a longduration”) or verbally estimate the duration (“that was a 4 sduration”). In discrimination a comparison is made between two durations(“the second stimulus was longer than the first”). Finally, inreproduction a response is made that bears some relation with thestimulus (e.g. only responses that are as long or longer than thestimulus are correct).

Time perception is a particularly effective measure for testingcognitive deficits in ADHD individuals. For example, Conners & Levin(1996) showed that ADHD adults improve in measures of attention andtiming with the administration of nicotine. Nicotine, like thepsychostimulants methylphenidate and d-amphetamine, acts as an indirectdopamine agonist and improves attention and arousal. Studies indicatethat adults and adolescents with ADHD smoke much more frequently thannormal individuals or those with other psychiatric conditions, perhapsas a form of self-medication for ADHD symptoms. The results indicatedthat there was a significant clinician-rated global improvement,self-rated vigor and concentration, and improved performance onchronometric measures of attention and timing accuracy, and side effectswere minimal (Conners & Levin, supra).

Eltoprazine hydrochloride [1-(2,3-dihyro-1,4-benzodioxin-5-yl)piperazine hydrochloride], a phenylpiperazine derivative, was originallydeveloped as a “serenic.” “Serenics” are drugs developed for theselective treatment of aggressive behavior, without negatively affectinggeneral functioning or motor abilities, and which demonstrate minimalside effects. Thus, eltoprazine was developed to treat and manageinappropriate aggression with high specificity. While unsuccessful inclinical trials, eltoprazine did prove to be clinically safe (de Koninget al., Int. Clin. Psychopharmacol., 9:187-194, 1994).

It has been hypothesized that the mechanism of action for eltoprazine inaggression is associated with activation of central serotonergic(5-hydroxytryptophan, 5-HT) systems (Schipper, J. et al., DrugMetabolism & Drug Interactions, 8:85-114, 1990). In adults, central 5-HTneurotransmission is inversely correlated with aggression: diminished5-HT function is associated with increased aggression. However, such arelationship is reported to be non-existent in children, including thosehaving ADHD (Schulz et al., Psychiatry Res., 101:1-10, 2001).

At present, seven main 5-HT receptor classes have been identified:5-HT₁, 5-HT₂, 5-HT₃, 5-HT₄, 5-HT₅, 5-HT₆ and 5-HT₇. Radioligand bindingstudies have revealed at least five subtypes of the 5-HT₁ receptor (1A,1B, 1D, 1E and 1F). Because the 5-HT_(1B) receptors are present in thehippocampal formation, it has been suggested that a potential role forthese receptors is the modulation of memory processes (Malleret, J.Neurosci., 19:6157-68, 1999). Serotonin inhibits acetylcholine releasethrough 5-HT_(1B) receptors located on septal terminals in thehippocampus (Maura and Raiteri, Eur. J. Pharmacol., 129:333-337, 1986)and glutamate release in the dorsal subiculum through 5-HT_(1B)receptors located on CA1 pyramidal neuron terminals (Aït Amara et al.,Brain Res. Bulletin, 38(1):17-23, 1995). Stimulation of the hippocampalreceptors in rats resulted in impaired spatial learning tasks andneophobic reactions in an object exploration task (Buhot and Naili,Hippocampus, 5:198-208, 1995). Thus, the blockade of 5-HT_(1B) receptorspotentially affects attention and emotion and positively affectslearning and memory processes (Buhot et al., supra). Therefore,5-HT_(1B) agonists would be predicted not to enhance attention orcognitive function.

The binding profile of eltoprazine, together with the direct bindingdata obtained with [³H] eltoprazine, shows the compound to be aselective 5-HT₁ ligand (selective with respect to all receptors otherthan 5-HT₁). Eltoprazine's binding affinity for the various 5-HTreceptor subtypes closely resembles serotonin except for the relativelylow affinity for the 5-HT_(1D) receptor with roughly equipotent affinityfor the 5-HT_(1A), 5-HT_(1B), and 5-HT_(2C) receptors (Schipper, J. etal., supra). Eltoprazine acts as a mixed 5-HT_(1A/B) receptor agonist.Eltoprazine has no relevant affinity for dopamine receptors (i.e.,K_(i)>1 μM, Schipper et al., supra). Among the 5-HT receptors, the5-HT_(1B) is located as an autoreceptor on axon terminals and isresponsible for inhibiting neurotransmitter release, whereas it is alsolocated postsynaptically as a heteroreceptor on axons and terminals ofnon-serotonergic neurons inhibiting their activity.

Pharmacokinetic studies have indicated that eltoprazine HCl is very wellabsorbed, with an absolute bioavailability of about 95%. The maximumplasma concentration of eltoprazine is attained within 1-4 hours afteradministration, followed by a decrease in plasma concentration with aterminal half-life of 7-9 hours. The cumulative renal excretion ofunchanged eltoprazine is about 40%. The plasma elimination half-liferanges between 5-12 hours. Eltoprazine plasma concentrations increase ina linear dose-dependent manner (De Vries et al., Clinical Pharmacology,41:485-488, 1991).

SUMMARY OF INVENTION

This invention relates to methods and compositions useful for treatingADHD in humans and the behaviors associated therewith. The compounds foruse in the invention are believed to be effective in the treatment ofADHD and exhibit reduced side effects and are not expected to have abusepotential, as compared to other available therapeutics.

Treatment of ADHD according to this invention may be used to reduce oneor more of any of the diagnostic criteria associated with ADHD. In apreferred embodiment of this invention, eltoprazine is administered toindividuals to provide treatment of symptoms associated with ADHD. Oneobject of the invention is to provide a method for treating ADHD byadministering to an individual a therapeutically effective amount of acompound of formula 1

wherein

-   -   R₁ is hydrogen, alkyl, cycloalkyl, optionally esterified        hydroxyalkyl, alkoxyalkyl, optionally substituted phenyl or        heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, acyl,        alkoxycarbonyl, aminocarbonyl, alkyl- or dialkyl-aminocarbonyl,        nitro, amino, alkyl- or dialkyl-amino, acylamino,        alkylsulfonylamino, arylamino, cyano, halogen, trifluoromethyl,        trifluoromethoxy, optionally esterified hydroxyl, alkyl- or        amino-sulphonyl or -sulphinyl, alkyl- or dialkyl-aminosulphonyl        or -sulphinyl, and p has the value 0-3;    -   R₂ and R′₂ are independently hydrogen or an alkyl group and n        and q can have the value 0 or 1;    -   R₃ may have the same meaning as R₁, or is alkylidene, an oxo or        thioxogroup, and m has the value 0-2;    -   A forms, with the two carbon atoms of the phenyl group, an        optionally entirely or partly unsaturated cyclic group having        5-7 atoms in the ring, which comprises 1-3 hetero atoms from the        group O, S, and N, with the proviso that the sum of the number        of oxygen and sulphur atoms is at most 2; and        wherein    -   the compound may be a racemate or a single diastereomer or        enantiomer;    -   or a pharmaceutically acceptable acid addition salt thereof.

In addition, the present invention provides a method for improvingcognitive function associated with ADHD.

Another object of the invention is to provide pharmaceuticalcompositions for the treatment of inattention and/orhyperactivity-impulsivity associated with ADHD that have reduced sideeffects as compared to other available treatments.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIGS. 1 A-C—Graphs depict the relative response rate of C57BL/6J mice inthe Peak Procedure (30 second reinforcement interval) afteradministration of 1, 2, or 4 mg/kg of d-amphetamine. * p<0.05; **p<0.01; *** p<0.001.

FIGS. 2 A-C—Graphs depict the relative response rate of C3H mice in thePeak Procedure (30 second reinforcement interval) after administrationof 1, 2, or 4 mg/kg of eltoprazine. ** p<0.01; *** p<0.001.

FIG. 3-Graphs depict the relative response rate of C3H mice in the PeakProcedure (30 second reinforcement interval) after administration of lowdoses, 0.1 and 0.9 mg/kg, of eltoprazine. * p<0.05; ** p<0.01; ***p<0.001.

FIG. 4-Graph depicts the effect of 4 mg/kg amphetamine on locomotoractivity in coloboma mutant and wild-type mice, as measured by totaldistance traveled in a fixed time period. * p<0.05; ** p<0.01.

FIGS. 5 A-B—Graphs depict the effect of 0.5 mg/kg eltoprazine onlocomotor activity in coloboma mutant and wild-type mice; 5A: distancetraveled per 5 minute block of the behavioral session; insert showstotal distance traveled in the session; 5B: depicts the frequency ofzone crossings per 5 minute block of the behavioral session; insertshows total number of crossings in the session. * p<0.05 compared toeltoprazine.

FIGS. 6 A-D—Graphs depict the effect of eltoprazine on exploratorypreference in coloboma mutant and wild-type mice; 6A: percent (%) timespent in the center of open field arena; 6B: distance traveled in thecenter of open field arena as a percent (%) of total distance traveledin entire arena; 6C: frequency of rearing in the center of the arena per5 minute block of the behavioral session; 6D: frequency of rearing inthe periphery of the arena per 5 minute block of the behavioral session.

FIG. 7-Graph depicts the effect of 5-HT_(1B) receptor agonist CP 94253,0.5 mg/kg, on locomotor activity in coloboma mutant and wild-type mice.

FIG. 8-Graph depicts the effect of 5-HT_(1B) receptor agonist CP 94253,0.3, 1 or 3 mg/kg, i.p., on response rate of C3H mice in Peak Procedure(30 second reinforcement interval).

FIGS. 9 A-B—Graphs depict acquisition of operant responding in theautoshaping paradigm by wild-type, 5-HT_(1B) knockout mice (1BKO), and5-HT_(1A) knockout (1AKO) mice. Time in minutes needed to reachcriterion (9A) during autoshaping, and number of nose pokes made permin±SEM (9B). Data are depicted as means±SEM. * p<0.05 compared towild-type.

FIGS. 10 A-C—Graphs depict acquisition of a differential reinforcementof low rates-36 second (DRL-36s) schedule by 5-HT_(1B) receptor knockoutmice compared to wild-type mice; 10A: comparison of the number ofresponses made over the course of training session; 10B: comparison ofthe number of reinforcements received over course of training session;10C: comparison of peak wait time to respond over course of trainingsession.

FIGS. 11 A-C—Graphs depict the inter-response time (IRT) histograms ofperformance under stable DRL-36 sec responding in WT (11A), 1AKO (11B)and 1BKO mice (11C). Frequency, as decimal fraction of whole, is plottedagainst IRT, in 3 second bins, with 36 seconds marked as the target forreinforcement. The bimodal IRT distribution of the DRL-36 sec isdepicted, with one mode at short IRT durations (IRT<3 sec, grey bar onleft) indicating bursting and a second mode at longer IRT durationsindicating pausing (IRT>3 sec, white bars). The connected dots indicatethe corresponding negative exponential which depicts random performance.Peak location (PkL or the “median” of the curve) was 35.2, 22.4, and 34sec. for wild-type, 1BKO, and 1AKO mice, respectively.

FIGS. 12 A-B—Graphs depict the effect of d-amphetamine (2, 4 or 8 mg/kg,i.p.) on 12A: the number of reinforcements and 12B: the response rate ofwild-type (WT) and 5-HT_(1B) knockout (1BKO) mice trained on afixed-rate 5 (FR5) DRL-36s schedule. * p<0.05 compared to vehicle;+p<0.05 WT vs. 1BKO.

FIGS. 13 A-B—Graphs depict effect of eltoprazine (0.25, 0.5 or 1 mg/kg,i.p.) on 13A: the number of reinforcements and 13B: the response rate ofwild-type (WT) and 5-HT_(1B) knockout (1BKO) mice with a history offixed-ratio 5 second (FR5) responding, challenged on a DRL-36sschedule. * p<0.05 compared to vehicle.

FIG. 14-Graph depicts the effect of eltoprazine (0.1 mg/kg, i.p.) onpercent change in basal DA and 5-HT outflow in dorsal striatum of awake,freely moving wild-type mice. DA and 5-HT release was measured by invivo microdialysis coupled to HPLC-ECD. DA or 5-HT levels are expressedas percentages of basal levels±SEM. Dialysate was sampled every 20minutes; time of drug administration is indicated by the arrow.

FIG. 15-Graph depicts the effect of local administration of the5-HT_(1B) receptor agonist CP 93129 (0.5 μM) on percent change in basal5-HT outflow in dorsal striatum of awake, freely moving wild-type and5-HT_(1B) knockout (1BKO) mice. 5-HT release was measured by in vivomicrodialysis coupled to HPLC-ECD. Dialysate was sampled every 20minutes; time of drug introduction through microdialysis probe isindicated by the solid black bar.

FIG. 16-Graph depicts the effect of local administration of the5-HT_(1B) receptor agonist CP 93129 (0.5 or 50 μM) on percent change inbasal DA outflow in dorsal striatum of awake, freely moving wild-typeand 5-HT_(1B) knockout (1BKO) mice. DA release was measured by in vivomicrodialysis coupled to HPLC-ECD. Dialysate was sampled every 20minutes; time of drug introduction through microdialysis probe isindicated by the solid black bar.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of treating ADHD in humans. Asused herein, ADHD is intended to comprise the distinct sets of symptomsassociated with the three subtypes defined in DSM-IV-TR, inattention,hyperactivity/impulsivity, or combined, which present in an individualas ADHD. Impulsivity associated with ADHD is present with othersymptoms, i.e., hyperactivity or hyperactivity and inattention.

ADHD of the predominantly inattentive type is diagnosed if six (or more)of the following symptoms of inattention (and fewer than six of thehyperactivity-impulsivity symptoms below) have persisted for at least 6months to a degree that is maladaptive and inconsistent withdevelopmental level. The inattention component of ADHD may include oneor more of the following symptoms: (a) often fails to give closeattention to details or makes careless mistakes in schoolwork, work, orother activities, (b) often has difficulty sustaining attention in tasksor play activities, (c) often does not seem to listen when spoken todirectly, (d) often does not follow through on instructions and fails tofinish school work, chores, or duties in the workplace (not due tooppositional behavior or failure to understand instructions), (e) oftenhas difficulty organizing tasks and activities, (f) often avoids,dislikes, or is reluctant to engage in tasks that require sustainedmental effort (such as schoolwork or homework), (g) often loses thingsnecessary for tasks or activities (e.g., toys, school assignments,pencils, books, or tools), (h) is often easily distracted by extraneousstimuli, and (i) is often forgetful in daily activities (DSM-IV-TR,supra).

ADHD of the predominantly hyperactive/impulsive type is diagnosed if six(or more) of the following symptoms of hyperactivity-impulsivity (andfewer than six of the inattention symptoms above) have persisted for atleast 6 months to a degree that is maladaptive and inconsistent withdevelopmental level. The hyperactivity component of ADHD may include oneor more of the following symptoms: (a) often fidgets with hands or feetor squirms in seat, (b) often leaves seat in classroom or in othersituations in which remaining seated is expected, (c) often runs aboutor climbs excessively in situations in which it is inappropriate (inadolescents or adults, may be limited to subjective feelings ofrestlessness), (d) often has difficulty playing or engaging in leisureactivities quietly, (e) is often “on the go” or often acts as if “drivenby a motor,” and (f) often talks excessively. The impulsivity componentof ADHD may include one or more of the following symptoms: (g) oftenblurts out answers before questions have been completed, (h) often hasdifficulty awaiting turn, and (i) often interrupts or intrudes on others(e.g. butts into conversations or games) (DSM-IV-TR, supra).

The most common subtype of ADHD is the combined type, which comprisesall three sets of symptoms, inattention, hyperactivity and impulsivity.Combined-type ADHD is diagnosed if six (or more) symptoms of inattentionand six (or more) symptoms of hyperactivity/impulsivity have persistedfor at least 6 months (DSM-IV-TR, supra). ADHD of the combined type, aswell as the inattentive and hyperactive/impulsive subtypes, may betreated according to this invention.

Unlike traditional therapeutics, which have the potential to be abusedand/or have undesirable side effects, the present invention is notexpected to have the abuse potential of psychostimulants, the mostwidely prescribed current pharmacological treatment, and may have a sideeffect profile distinct from other types of pharmacologic therapeutics.Therefore, an advantage of the method of ADHD treatment provided by thisinvention is that certain of the undesirable side effects may be reducedor avoided.

As discussed above, ADHD is diagnosed based on an individual possessingsymptoms in the symptom clusters inattentiveness, hyperactivity andimpulsiveness, as defined according to the DSM-IV-TR and recognized inthe art.

The compounds for use with this invention, preferably eltoprazine, maybe used to treat ADHD and/or the specific symptoms or variouscombinations of the constellation of symptoms associated with ADHD. Whensymptoms associated with ADHD are treated according to this invention,preferably at least two symptoms associated with ADHD are present, andwhen a symptom in the impulsivity cluster is present, then anothersymptom of ADHD in the hyperactivity or inattentiveness cluster is alsopresent.

Treatment of ADHD according to this invention is provided byadministering to an individual in need of treatment a therapeuticallyeffective amount of a compound of formula 1:

wherein

-   -   R₁ is hydrogen, alkyl, cycloalkyl, optionally esterified        hydroxyalkyl, alkoxyalkyl, optionally substituted phenyl or        heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, acyl,        alkoxycarbonyl, aminocarbonyl, alkyl- or dialkyl-aminocarbonyl,        nitro, amino, alkyl- or dialkyl-amino, acylamino,        alkylsulfonylamino, arylamino, cyano, halogen, trifluoromethyl,        trifluoromethoxy, optionally esterified hydroxyl, alkyl- or        amino-sulphonyl or -sulphinyl, alkyl- or dialkyl-aminosulphonyl        or -sulphinyl, and p has the value 0-3;    -   R₂ and R′₂ are independently hydrogen or an alkyl group, and n        and q can have the value 0 or 1;    -   R₃ may have the same meaning as R₁, or is alkylidene, an oxo or        thioxogroup, and m has the value 0-2;    -   A forms, with the two carbon atoms of the phenyl group, an        optionally entirely or partly unsaturated cyclic group having        5-7 atoms in the ring, which comprises 1-3 hetero atoms from the        group O, S, and N, with the proviso that the sum of the number        of oxygen and sulphur atoms is at most 2.

Unless otherwise defined, an alkyl is 1-10 carbons, aryl is 6-10carbons, and cycloalkyl is 3-10 carbons.

When a halogen, R₁ is preferably fluoro, chloro or bromo, and when analkyl group, R₁ is preferably a straight or branched, saturated orunsaturated group having 1-5 carbon atoms.

When an alkyl group, R₂ is preferably a methyl or ethyl group.

When a hydroxyalkyl group, R₃ preferably comprises 1-3 carbon atoms.

When R₁ or R₃ is an esterified hydroxyl group or hydroxylalkyl group,the ester group preferably has the formula O—CO—R₄ or —CS—R₄ in which R₄is alkyl, aralkyl, aryl, heteroaryl, hetero aralkyl, wherein the alkylgroup may be branched or unbranched, and the (hetero) aryl part mayoptionally be substituted, or R₄ may be an alkoxy, heteroalkoxy ordialkylamino group, in which the two alkyl groups can form ahetero-cyclic ring with the nitrogen atom.

When R₁ or R₃ is an etherified hydroxyl group or hydroxyalkyl group, theether group preferably has the formula —O—R₅, wherein R₅ is a straight,branched or cyclic alkyl group having 1-5 C-atoms, or an alkoxyalkylgroup having 1 or 2 C-atoms in both the alkoxy part and in the alkylpart thereof.

Eltoprazine (1-(2,3-dihydro-1,4-benzodioxanyl-5-yl) piperazine) isparticularly preferred for use with this invention: R₁, R₂, R′₂ and R₃are hydrogen and A, together with the phenyl ring to which it isattached, forms a 2,3-dihydro-1, 4-benzodioxin, C₁₂H₁₆N₂O₂; orpharmaceutically acceptable salts thereof, preferably HCl. Anotherpreferred compound that may be useful for this invention is batoprazine,(8-(1-piperazine)-2H-1-benzopyran-2-one). This invention also includesthe use of prodrugs of the compounds of formula 1, specificallyderivatives of the compounds of formula 1 that are inactive but areconverted to an active form in the body following administration.

The compounds described above including eltoprazine and their method ofsynthesis are known in the art and are described in U.S. Pat. No.4,833,142; U.S. Pat. No. 5,424,313; European Patent No. 189,612; andEuropean Patent No. 138,280, which are incorporated herein by referencein their entirety.

ADHD and/or symptoms associated with ADHD are treated according to thisinvention by administering therapeutic dosages of compounds according toformula 1.

Eltoprazine's utility for treating ADHD and symptoms associated withADHD, is based on the surprising discovery disclosed herein thateltoprazine shares certain activity profiles with other compounds knownto be useful for treating such conditions. Amphetamines enhancemonoaminergic transmission; however, their mechanism of action in ADHDis still the subject of much speculation. Without being bound by theory,one possible mechanism is the enhancement of dopamine release in thoseareas of the brain that are involved in attentional mechanisms, such asthe frontal cortex, however, such a model seems to be overly simplisticand incomplete. (Nestler, Hyman, & Malenka, Molecular Neuropharmacology:A Foundation for Clinical Neuroscience, McGraw Hill, 2001). Psychoactivesubstances such as amphetamines typically show a U-shape curve, with lowdoses being cognitive enhancers, and high doses being disruptive ofcognitive performance.

The mechanism underlying these U-shape curves is poorly understood, withone possibility being the differential action on pre- and postsynapticdopamine D2 receptors. It is possible that low doses preferentiallyaffect the post- (or pre-) synaptic receptors, and that only higherdoses affect both types. The differential action could be the result ofdifferent binding characteristics (due to subtle changes in thereceptors), or to differences in the amount of receptor reserve (wherehigh receptor reserve results in a stronger effect). This dual pre- andpostsynaptic action of dopamine (and of dopamine agonists) is mimickedin the serotonergic system, in which the 5-HT_(1A) and 5-HT_(1B)receptors exist as both autoreceptors (presynaptic) and heteroreceptors(postsynaptic) and have opposite effects. Presynaptic action typicallyresults in a reduction of neurotransmitter release (and less activationof target receptors), whereas postsynaptic action results in enhancedactivation of target receptors.

Although the main target of amphetamine-like drugs (and of bupropion,one antidepressant used for ADHD when adverse reactions prevent the useof psychostimulants) is the dopaminergic system, strong interactionsbetween dopamine and serotonin are known. As a result, drugs that affectthe serotonin system will very likely have secondary effects in thedopaminergic system. Moreover, serotonergic drugs that have a dual pre-and postsynaptic action would be expected to show U-shaped responses.Thus, a drug which acts as a cognitive enhancer at low doses, anddisrupts performance at high doses, may be a drug that mimicsamphetamine-like effects, and therefore may be of value in the treatmentof ADHD.

The dose of the compound used in treating ADHD in accordance with thisinvention will vary in the usual way with the seriousness of thedisorder, the weight, and metabolic health of the individual in need oftreatment. The preferred initial dose for the general patient populationwill be determined by routine dose-ranging studies, as are conducted,for example, during clinical trials. Therapeutically effective doses forindividual patients may be determined, by titrating the amount of druggiven to the individual to arrive at the desired therapeutic orprophylactic effect, while minimizing side effects. A preferred initialdose for this compound, may be estimated to be between about 0.1 mg/dayand 100 mg/day. More preferably, the initial dose is estimated to bebetween 0.1 mg/day and 30 mg/day. Even more preferred, the initial doseis estimated to be between 0.1 mg/day and 10 mg/day.

To achieve a therapeutic effect for ADHD and symptoms thereof, thepreferred plasma concentration of the compounds for use with thisinvention is between about 0.06 ng/ml and about 200 ng/ml in a human.The preferred plasma concentration of eltoprazine for use with thisinvention is between about 0.2 ng/ml and about 65 ng/ml in a human.

Administration of the compounds of this invention may be by any methodused for administering therapeutics, such as for example oral,parenteral, intravenous, intramuscular, subcutaneous, or rectaladministration.

In addition to comprising the therapeutic compounds for use in thisinvention, especially eltoprazine [1-(2,3-dihyro-1,4-benzodioxin-5-yl)piperazine] or pharmaceutically acceptable salts (preferably HCl in thecase of eltoprazine) or pro-drug thereof, the pharmaceuticalcompositions for use with this invention may also comprise apharmaceutically acceptable carrier. Such carriers may compriseadditives, such as preservatives, excipients, fillers, wetting agents,binders, disintegrants, buffers may also be present in the compositionsof the invention. Suitable additives may be, for example magnesium andcalcium carbonates, carboxymethylcellulose, starches, sugars, gums,magnesium or calcium stearate, coloring or flavoring agents, and thelike. There exists a wide variety of pharmaceutically acceptableadditives for pharmaceutical dosage forms, and selection of appropriateadditives is a routine matter for those skilled in art of pharmaceuticalformulation.

The compositions may be in the form of tablets, capsules, powders,granules, lozenges, suppositories, reconstitutable powders, or liquidpreparations such as oral or sterile parenteral solutions orsuspensions.

In order to obtain consistency of administration it is preferred that acomposition of the invention is in the form of a unit dose. Unit doseforms for oral administration may be tablets, capsules, and the like,and may contain conventional excipients such as binding agents, forexample syrup, acacia, gelatin, sorbitol, tragacanth, orpolyvinylpyrrolidone; and carriers or fillers, for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine. Additivesmay include disintegrants, for example starch, polyvinylpyrrolidone,sodium starch glycolate or microcrystalline cellulose; preservatives,and pharmaceutically acceptable wetting agents such as sodium laurylsulphate.

In addition to unit dose forms, multi-dosage forms are also contemplatedto be within the scope of the invention. Delayed-release compositions,for example those prepared by employing slow-release coatings,micro-encapsulation, and/or slowly-dissolving polymer carriers, willalso be apparent to those skilled in the art, and are contemplated to bewithin the scope of the invention.

The solid oral compositions may be prepared by conventional methods ofblending, filling, tabletting or the like. Repeated blending operationsmay be used to distribute the active agent throughout those compositionsemploying large quantities of fillers. Such operations are conventionalin the art. The tablets may be coated according to methods well known innormal pharmaceutical practice, for example with an enteric coating.

Oral liquid preparations may be in the form of, for example, emulsions,syrups, or elixirs, or may be presented as a dry product forreconstitution with water or other suitable vehicle before use. Suchliquid preparations may contain conventional additives such assuspending agents, for example sorbitol syrup, methyl cellulose,gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminumstearate gel, and hydrogenated edible fats; emulsifying agents, forexample lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles(which may include edible oils), for example almond oil or fractionatedcoconut oil, oily esters such as esters of glycerine, propylene glycol,or ethyl alcohol; preservatives, for example methyl or propylp-hydroxybenzoate or sorbic acid; and if desired conventional flavoringor coloring agents.

For parenteral administration, fluid unit dosage forms are preparedutilizing the compound and a sterile vehicle, and, depending on theconcentration used, can be either suspended or dissolved in the vehicle.In preparing solutions the compound can be dissolved in water or salinefor injection and filter sterilized before filling into a suitable vialor ampoule and sealing. Advantageously, additives such as a localanaesthetic, preservative and buffering agent can be dissolved in thevehicle. Suitable buffering agents are, for example, phosphate andcitrate salts. To enhance the stability, the composition can be frozenafter filling into the vial and the water removed under vacuum.Parenteral suspensions are prepared in substantially the same manner,except that the compound is suspended in the vehicle instead of beingdissolved, and sterilization cannot be accomplished by filtration. Thecompound can be sterilized by conventional means, for example byexposure to radiation or ethylene oxide, before being suspended in thesterile vehicle. Advantageously, a surfactant or wetting agent isincluded in the composition to facilitate uniform distribution of thecompound.

The invention will be explained in more detail below by way of examples,which illustrate the effectiveness of prototypical compound eltoprazinein alleviating symptoms associated with ADHD.

EXAMPLE 1

The peak procedure is a behavioral model designed to assess an animal'sability to learn an appropriate time period in which to perform a taskand a time period in which the animal will be rewarded if the task isperformed. The model provides information concerning excitatory andinhibitory components of behavior, as subjects must respond to perform atask when appropriate and stop responding in an “empty trial” when timefor reward has elapsed and the reward has not been delivered. The taskis sensitive to conditions where there is a failure in inhibitorymechanisms, such as seems to be the case for ADHD (Pliszka et al., Biol.Psychiatry, 48:238-46, 2000).

In the peak procedure, mice are trained to work for food that isdelivered at the same time in each trial, but withdrawn in someunreinforced trials. Typically, the response rate increases up to amaximum around the reinforcement time, and then decreases to a lowtoward the end of the trial. The shape of the response rate indicateswhether the animal is sensitive to the time of reinforcement. To be ableto perform well in this model, the animals need to be able to learnseveral tasks. First, the animal must make an association between aresponse (lever pressing, nose poking or key pecking) and the deliveryof reward. Second, the animal must be able to perceive and remembertime. Third, the animal must act on its remembered time by starting andthen stopping or inhibiting the response. Fourth, the animal must beable to compare the elapsed time in the trials with its remembered timeto reinforcement. In each trial the time clock is reset, and the animalmust reset its internal “counter,” i.e., at the beginning of each trialanimals should start “timing” the trial time from zero. The ability toperform this task depends on the animal's working memory. Starting theinternal clock at the beginning of the trial requires that the animalpays attention to the trial start time, which could be in the form of avisual signal, or as reported herein, the introduction of a lever intothe experimental chamber. Failure to attend resulted in highervariability and a loss of accuracy during trial performance. Accuracy ismeasured by looking at the shape of the response function; therefore, ifthe response function is sharper and centered on the reinforcement timeit supports a conclusion that attentional processes have beenheightened.

Mice were food deprived to 85-90% of their free-feeding body weight bysupplementing food earned during experimental sessions with a measuredamount after the session had ended. For the amphetamine dose responsecurve, C57BL/6J mice were used (n=14). For the eltoprazine study, C3Hmice were used (n=14). Once deprived, animals were trained tolever-press in an operant box (Med Associates) using ultrasensivelevers. During the training, food was delivered after any one press ofthe lever. Once lever-pressing was robust (about 1 week), a fixedinterval of 10 seconds was introduced between the beginning of a trial(when the lever is introduced into the chamber) and the reinforcedresponse. All premature responses had no programmed consequences. Afterone week the fixed interval was increased to 30 seconds and animals weretrained on this new fixed interval until response curves were stable.The last phase of training included empty or “peak” trials in whichreinforcement was withdrawn and the trial continued for 3 times thefixed interval.

Once the performance during peak trials was stable, a dose responsestudy was initiated. Drug injections were delivered 30 minutes prior tothe session. Doses were scheduled on Monday, Wednesday and Friday, withTuesday and Thursday being normal sessions without a drug. Eltoprazinedose responses were done on the same mice with at least 1 week ofwashout period. During this time all responses were unreinforced.Responses during these peak trials were recorded and transformed into arelative responding measure by dividing the number of responses in each5 minute bin by the maximum response rate at any time interval in thattrial. After relative responses had been calculated for each trial, anAnalysis of Variance (ANOVA) with trial time and dose as within factorswas performed. Significant interactions were followed up by plannedpair-wise comparisons between the saline response and the correspondingdrug dose response.

In subjects with problems of inhibition and response control, it will bebeneficial to find a drug that improves performance by sharpening theresponse curve and providing the subject with greater control over thestart and stop time for response. The experiments with mice and thetiming procedure were designed to maximize the chance of finding drugsthat improve performance. Amphetamine was tested in low to moderatelyhigh doses. Amphetamine, a drug of abuse, is used by humans as acognitive enhancer at low doses, and as a recreational drug (thatresults in a “high” state) at much higher doses (more than five timesthe cognitive enhancing dose).

FIG. 1 shows the response pattern obtained with d-amphetamine. At thelower doses, 1 and 2 mg/kg, the amphetamine curve demonstrates a higherpeak in the curve followed by a rapid decrease, relative to saline(FIGS. 1A, 1B). Conversely, at the higher 4 mg/kg dose, the amphetaminecurve does not peak as high as the lower dose and the curve is flatter(FIG. 1C). Times at which pairwise comparisons between saline andamphetamine reached significance are indicated on the graphs. ANOVArevealed a significant dose×trial time interaction, p<0.001.

The lowest two doses acted as cognitive enhancers as they sharpened thecurve. The highest dose disrupted performance, flattening the curve.Improved performance is demonstrated by a sharp peak in the curvefollowed by a rapid decrease. A less marked peak in the curve followedby a flattening is indicative of deteriorated performance. Cognitiveenhancement, as used herein, refers to heightened attentional processes.

EXAMPLE 2

The following results were obtained using the methods described inExample 1. Eltoprazine was investigated using a very wide dose range:0.1-4 mg/kg. FIG. 2 demonstrates decreased cognitive enhancement athigher doses of eltoprazine. At 1, 2, and 4 mg/kg eltoprazine, theperformance curves were flatter than the saline curve (FIGS. 2A, 2B, and2C, respectively). ANOVA revealed a significant dose main effect,p<0.001, and a significant dose×trial time interaction, p<0.01.

At lower doses of eltoprazine, however, cognitive enhancement wasobserved, as illustrated in FIG. 3. In two separate studies of 0.1 and0.9 mg/kg eltoprazine, the peaks of the response curves are higher andthe curves are sharper. Times at which pairwise comparisons betweensaline and amphetamine reached significance are indicated on the graphs.ANOVA revealed a significant dose main effect in one study, p<0.001, anda significant dose×trial time interaction in both studies, p<0.01. Inconclusion, eltoprazine at low doses, may act as a cognitive enhancerand is expected to be useful in the treatment of ADHD and associatedsymptoms thereof.

EXAMPLE 3

The coloboma (Cm) mutant mouse has been proposed as a rodent model forADHD (for review, see Wilson, Neurosci. Biobehav. Rev., 24:51-57, 2000).The rationale for this proposal is three fold: first, Cm mutants(heterozygote) exhibit elevated spontaneous locomotor hyperactivitywhich averages three to four times the activity of wild-type littermates(Hess et al., J. Neurosci., 12:2865-2874, 1992; Hess et al., J.Neurosci., 16:3104-3111, 1996); second, this Cm mutation-associatedhyperactivity can be ameliorated by low and moderate doses (2-16 mg/kg)of D-amphetamine (Hess et al., 1996, supra), a psychostimulant commonlyprescribed to treat ADHD; and lastly, Cm mutant mice exhibit delays inachieving complex neurodevelopmental milestones in behavior (Heyser etal., Brain Res. Dev. Brain Res., 89:264-269, 1995) and deficits inhippocampal physiology and learning performance (Steffensen et al.,Synapse, 22:281-289, 1996; Raber et al., J. Neurochem., 68:176-186,1997) which may correspond to impairments seen in ADHD.

The genetic defects associated with Cm mutant mice include a deletion ofthe gene Snap (Hess et al., 1992, supra; Hess et al., Genomics,21:257-261, 1994). Snap encodes SNAP-25, which is a key component of thesynaptic vesicle docking and fusion complex required for regulatedsynaptic transmission. As a result, Cm mutant animals show markeddeficits in Ca²⁺-dependent dopamine release (Raber et al., supra). Thishypofunctioning DA system, which may involve meso-cortical, meso-limbic,as well as nigro-striatal circuitries has been suggested as a possiblemechanism underlying hyperactivity associated with Cm mutation(Sagvolden, et al., Behav. Brain Res., 94:61-71, 1998; Sagvolden andSergeant, Behav. Brain Res., 94:1-10, 1998).

Amphetamine, but not methylphenidate, normalizes the hyperactivity in Cmmutant mice; in both control and Cm mutants, methylphenidate increaseslocomotor activity in a dose-dependent manner (Hess et al., 1996,supra). The differential effect of these two ADHD medicaments, whichboth act at the presynaptic terminal, has been attributed to thediffering mechanisms of action of increasing synaptic DA concentrations(Hess et al., 1996, supra).

It has now been surprisingly found that eltoprazine, a 5-HT_(1A/1B)receptor agonist, produces an amphetamine-like effect on hyperactivityin coloboma mice.

Animals

Heterozygote coloboma mice were originally purchased from The JacksonLaboratory (Bar Harbor, Me.) and were bred and maintained in our colony.In the current study, 20 mutant mice and 25 wild-type littermates, allaged 8 to 10 weeks, were used. Animals were divided into 4 groups:mutant/drug-treatment (n=11), mutant/vehicle-control (n=9),wild-type/drug-treatment (n=13), wild-type/vehicle-control (n=12). Ageand gender were balanced among groups. All animals were housed aslittermates (2-4 mice per cage) and were maintained on ad libitum foodand water with a 12 hr light/dark cycle.

Behavioral Testing

Open-field testing was performed under normal lighting conditions. Micewere brought into the experimental room and allowed at least 1 hr ofacclimatization. Thirty minutes prior to testing, animals received anip. injection of either d-amphetamine (4 mg/kg), eltoprazine (0.5 mg/kg)or saline. The mice were then placed in the activity monitor arenas(27×27×20 cm, Med Associates). Four animals of matching genotype andtreatment were tested at one time. Each testing session lasted 40minutes, after which animals were returned to their home cages. Anautomated infrared beam array system measured locomotor activity (totaldistance traveled) and number of center entries (zone crossings).

Results

The data reveal a significant genotypic effect on parameters ofhyperactivity that is reduced by eltoprazine treatment. Coloboma mutantmice are hyperactive relative to wild-type mice, as measured byincreased locomotor activity. A genotypic effect on total ambulatorydistance is depicted in FIGS. 4 and 5A (inset) and a genotypic effect ontotal crossed zones is depicted in FIG. 5B (inset). FIG. 4 and inset ofFIG. 5A illustrate that saline-treated mutant Cm mice traveled roughlythree-six times further in distance than did their saline-treatedwild-type littermates (18,386±6387 vs. 3116±338 cm, FIG. 4; 17725±6636vs. 6288±1565 cm, FIG. 5A), the scale of which is consistent withpreviously reported findings (Hess et. al., 1992, 1996 supra). Analysisof variance (ANOVA) revealed that this genotypic effect on totalambulatory distance was significant (F_((1,41))=6.798, p=0.0127) (FIG.5A). FIG. 5B illustrates that saline-treated mutant mice crossed zonesmore frequently than did their saline-treated wild-type littermates.ANOVA revealed this genotype effect on total crossed zones also issignificant (F_((1,41))=7.577, p=0.0088).

The genotype-related difference in locomotion was, however, largely andsignificantly diminished in eltoprazine-treated animals and reversed inamphetamine-treated animals.

Administration of amphetamine, 4 mg/kg, to wild-type mice had astimulatory effect, significantly increasing total distance traveled, asillustrated in FIG. 4 (3116±338 vs. 11657±2370 cm; ANOVA F(1,15)=11.276,p=0.0043). By contrast, the same dose of amphetamine administered to Cmmutant mice significantly decreased total distance traveled relative tosaline-treated Cm mutants (18386±6387 vs. 5966±1938 cm; ANOVAF(1,11)=5.355, p=0.0459) to within the range of saline-treated wild-typemice. Amphetamine effectively normalized the hyperactive locomotorbehavior of the coloboma mutant mice, significantly decreasinglocomotion in the Cm mutant mice. ANOVA revealed a significanttreatment×genotype interaction (F(1,25)=11.038, p=0.0027).

Eltoprazine did not influence the locomotor activity of wild-type mice,however, the effects of eltoprazine on Cm mutant mice were surprisinglysimilar to amphetamine. Indeed, administration of eltoprazine (0.5mg/kg, i.p.) reduced the total ambulatory distance of the Cm mutants to8641±1811, more than 50% reduction from that of saline-treated Cmmutants, as illustrated in FIG. 5A. Likewise, as depicted in FIG. 5B,eltoprazine decreased the number of zone crossings in Cm mutants towithin the range of saline-treated wild-type mice. Notably, eltoprazineonly marginally affected locomotion in wild-type animals, as measured bydistance traveled or zones crossed. This differential drug effect madethe locomotor activity of eltoprazine-treated mutant and wild-typeanimals indistinguishable from that of saline-treated wild-type animals.In other words, eltoprazine effectively normalized the hyperactivityassociated with the Cm mutation.

Eltoprazine has been tested in a variety of species, including human,over a broad range of doses, and the overall safety and tolerance of thecompound are good (de Koning et al., supra). Importantly, no sedativeeffect of eltoprazine was observed at the dose used. Indeed, theactivity of eltoprazine-treated animals is comparable to that of normalanimals in this study, which rules out the possibility that the calming(i.e., anti-hyperactive) effect of eltoprazine is due to a generalreduction in mobility.

It has previously been shown that the psychostimulant anti-ADHD agentsd-amphetamine, but not methylphenidate, reinstated normal locomotoractivity of the Cm mutants, suggesting an inconsistent effect ofpsychostimulants on this model of hyperactivity (Hess et. al., 1996,supra). However, the findings of this invention suggest that eltoprazinehas a specific regulatory role over hyperactivity. In summary,eltoprazine is acting like the anti-ADHD agent amphetamine in the Cmanimal model of ADHD, but lacks the adverse stimulant properties ofamphetamine observed in wild-type mice, demonstrating the therapeuticpotential and advantages of eltoprazine as an anti-ADHD agent.

EXAMPLE 4

Eltoprazine-induced normalization of locomotion in Cm mutant mice wasfound not to be associated with altered exploratory preference orfrequency of rearing. Although some researchers have argued thateltoprazine may enhance neophobia in rodents (Rodgers et. al., Behav.Pharmacol., 3:621-634, 1992; Griebel et al., Psychopharmacology (Berl),102:498-502, 1990), the doses (1.25 mg/kg or higher) and the tests used(elevated plus maze or light-dark box) in these studies were differentfrom those used in the present study.

Open-field testing was performed as described in Example 3. Rodents bynature are neophobic as measured by two parameters in the open-fieldtest including: exploratory preference for the periphery over center ofthe arena and the frequency of rearing. Eltoprazine did not alter eitherparameter in this study. As FIG. 6 illustrates, eltoprazine had noeffect on exploratory preference in either Cm or wild-type mice. Animalsof both treatment groups spent a comparable amount of time traveling inthe center and ambulated a similar distance (FIG. 6A, 6B); they alsoexhibited the same pattern of rearing behavior (FIG. 6C, 6D).

Taken together, Examples 3 and 4 indicate that eltoprazine selectivelydampens locomotor activity in the Cm mutant mouse without affectingother behaviors of the animal. Thus, the regulatory effect ofeltoprazine over Cm-induced hyperactivity is highly specific.

EXAMPLE 5

The primary targets for eltoprazine are reported to be 5-HT_(1A) and5-HT_(1B) receptors (see Schipper et al., supra). It surprisingly hasbeen discovered, however, that the effects of eltoprazine in alleviatingor normalizing symptoms associated with ADHD, such as hyperactivity, maybe mediated by mechanisms other than agonist action at 5-HT_(1B)receptors. If 5-HT_(1B) receptors are important in mediating theanti-ADHD effects of eltoprazine, a specific 5-HT_(1B) receptor agonistshould mimic the effects of eltoprazine in models of ADHD. 5-HT_(1B)receptor agonists were found not to produce the same effects aseltoprazine on locomotor activity.

The 5-HT_(1B) receptor agonist CP 94253 was tested in coloboma mutantmice in the open field test for locomotor activity using the methodsdescribed in Example 3. FIG. 7 demonstrates that, unlike eltoprazine, CP94253 fails to normalize the hyperactive behavior of coloboma mice. CP94253, at 0.5 mg/kg, did not decrease significantly the distancetraveled in coloboma mutants, but rather tended to increase distancetraveled. CP 94253 also had no effect on the locomotor activity ofwild-type mice. The effects of CP 94253 on locomotor activity incoloboma mice contrasts with the effects of both eltoprazine andamphetamine, surprisingly suggesting that the calming effects ofeltoprazine are mediated by mechanisms other than the 5-HT_(1B)receptor.

EXAMPLE 6

Another surprising discovery further indicates that the effects ofeltoprazine in alleviating or normalizing ADHD-associated behaviors suchas inattentiveness may be mediated by mechanisms other than agonistaction at 5-HT_(1B) receptors. 5-HT_(1B) receptor agonists were foundnot to produce the same effects as eltoprazine on the Peak Procedure.The 5-HT_(1B) receptor agonist, CP 94253, was tested in C3H mice in thepeak procedure using the methods described in Example 1. At dosesbetween 0.3 to 3.0 mg/kg, CP 94253 had no effect on timing in the peakprocedure in C3H mice, as depicted in FIG. 8. This contrasts with theeffects of both eltoprazine and amphetamine in this paradigm (seeExamples 1 and 2). These results provide further evidence thateltoprazine's therapeutic anti-ADHD effect of cognitive enhancementoccurs by a mechanism other than its known 5-HT_(1B) agonist activity.

EXAMPLE 7

To further determine whether or not 5-HT_(1B) receptors play a role inthe effectiveness of eltoprazine as an anti-ADHD therapeutic, mice inwhich the 5-HT_(1B) receptor was deleted by genetic knock out (Saudou etal., Science, 265:1875-1878, 1994) were tested on a delay ofreinforcement behavioral paradigm, a differential reinforcement of lowrate-36 second schedule (DRL-36s). Homozygous 5-HT_(1B) (1BKO) miceexhibit difficulty waiting for a specified time period to obtain areward (Brunner and Hen, supra). This difficulty provides a useful modelfor assessing the ability of a drug to alter animal behavior which mayreflect the hyperactivity-impulsivity symptoms of ADHD.

The hyperactivity-impulsivity of 1BKO mice was evaluated by comparingtheir behavior on a DRL-36s schedule with that of wild-type litter matesand homozygous 5-HT_(1A) knockout (1AKO) mice. In contrast to 1BKO mice,1AKO mice are hypoactive in the open field (Ramboz et al., Proc. Nat'lAcad. Sci., USA, 95:14476-81, 1998). 1AKO mice have been shown todisplay opposite behavioral phenotypes compared to 1BKO mice (Zhuang etal., Neuropsychopharmacology 21:52-60, 1999).

DRL schedules were originally developed and are used to screen putativeantidepressant drugs (O'Donnell and Seiden, J. Pharmacol. Exp. Ther.,224:80-88, 1983; Seiden et al., Psychopharmacology (Berl), 86:55-60,1985). Based on the ability to assess an animal's time perceptioncapacity and ability to obtain an award for learning to wait for aspecific time interval, measurements of DRL performance may be used tomeasure hyperactivity-impulsivity associated with ADHD (Monterosso andAinslie, Psychopharmacology, 146:339-47, 1999).

Animals

Male homozygote 5-HT_(1A) and 5-HT_(1B) receptor knockout and wild typemice were bred within the laboratory animal facilities of the UtrechtUniversity (GDL, Utrecht, The Netherlands). The breeding founders wereoriginally obtained from Dr. R. Hen (Columbia University, New York) andwere derived from established colonies from the 129/Sv strain (Saudou etal., supra; Ramboz et al., supra). Mice were generated by breedinghomozygote knockout and wild type mice with the same 129/Sv geneticbackground. Food consumption was restricted to keep the animals onapproximately 85% of their free-feeding weight.

Behavioral Testing

Experiments were conducted in eight identical mouse operant chambers(16×14×13 cm) with stainless steel grid floors (ENV-307M; Med AssociatesInc., Georgia, Vt., USA) housed in sound-insulating and ventilatedcubicles. Each chamber was equipped with two ultra-sensitive retractablelevers, one on each side of a food cup containing photocells to registernose poke behavior and in which a pellet dispenser delivered 20 mg foodpellets (Formula A/I, P.J. Noyes Company Inc., Lancaster, N.H., USA). Ared house light (50 lux) was located in the center of the wall oppositeto the food cup and levers. Stimulus lights were located above eachlever and above the food cup. Experimental sessions were controlled anddata were recorded by a computer.

The operant conditioning procedures used were a modification of thosedescribed by De Bruin et al. for rats (De Bruin et al., Prog. Brain Res.126:103-113, 2000), which is incorporated herein by reference. Threephases of conditioning were conducted: autoshaping, acquisition andreversal learning, and extinction. In autoshaping procedure, animals(n=8 per genotype) learned to lever-press for food under a fixed-ratio 1(FR1) schedule of reinforcement on both the left and right lever. At thebeginning of each trial, a stimulus light was illuminated on either theright or left, and the corresponding lever was inserted into thechamber. Pressing the lever below the stimulus light resulted in theimmediate delivery of a reinforcer signaled by the illumination of thestimulus light above the food cup, after which the stimulus light abovethe lever was extinguished and the lever retracted. Alternatively, whenthe lever was not pressed, after 60 sec the stimulus light wasextinguished and the lever retracted, without the delivery of a foodpellet. In either case, after a nose poke response into the food cup ora 30 sec time out period, a new trial started with an inter-trialinterval ranging from 5 to 25 sec (mean 15 sec). Criterion was reachedwhen animals earned in total 15 reinforcements during autoshapingsessions.

In acquisition and reversal, two levers are introduced into the chamberwithout illuminating stimulus lights. A session consisted of 50 trials,and mice were subjected to discrimination learning, i.e. only one of twoavailable levers was reinforced. When acquisition of discriminationlearning was fully mastered (criterion: accuracy between 95 and 100%correct lever presses), the task demands were changed, in that the otherlever was reinforced until criterion was reached. The extinction phasewas identical to acquisition and reversal but there was noreinforcement.

Mice trained on DRL procedure first received operant conditioning.DRL-36s task was adapted from procedures used in rats (O'Donnell andSeiden, supra). Briefly, mice first learned to respond for food under aDRL 6 sec schedule, which means that mice had to wait at least 6 secondsbetween successive lever presses in order to obtain a food reward.Subsequently the schedule requirement was increased every session insteps of 6 sec to 36 sec (DRL 36 sec). Each session started with theillumination of the house light, the stimulus light above the food cupand the presentation of the left lever for the duration of the session.Pressing the lever resulted in delivery of a food pellet when theinter-response time was longer than the required DRL time. If the mousepresses too soon, no reinforcement is given, the clock is reset to zero,and a new 36-sec waiting period starts. In this way, the DRL-36sschedule task primarily measures waiting strategies which reflecthyperactivity-impulsivity behaviors associated with ADHD. Animals weretrained until performances on the DRL 36 sec schedule had stabilized(approximately 25 sessions). All training sessions lasted 60 min andwere conducted 5 days per week from Monday-Friday. The total number oflever presses (responses), the total number of reinforcements, and theinter-response times (IRT) are recorded.

Data were analyzed as described elsewhere (Richards et al., J. Exp.Anal. Behav. 60:361-385, 1993; Sabol et al., Psychopharmacology121:57-65, 1995). The inter-response time (IRT) analysis included threemeasures for the characterization of DRL 36 sec IRT distributions: peakarea, peak location and burst ratio. Only peak area (PkA) and peaklocation (PkL) are presented. The PkA measure is the area of theobtained IRT distribution of a mouse above the corresponding negativeexponential, a computation based on mean of obtained IRT durationsexcluding the burst component of IRT<3 sec (see Richards et al., supra,for details). The largest possible PkA value (1.0) only occurs if allthe obtained IRT distributions had exactly the same value, whereas thesmallest PkA value (0) indicates that the obtained IRT distributions andcorresponding negative exponential are identical. Thus, decreases in PkAindicate that the mouse's IRT distribution becomes more similar torandom performance indicating loss of schedule control. PkL iscalculated as the median of the area of the obtained IRT distributionabove the corresponding negative exponential.

Results

The autoshaping behavior of the operant conditioning procedure for wildtype, 1BKO and 1AKO mice is depicted in FIG. 9. The time needed to reachcriterion (FIG. 9A) was different between genotypes (ANOVAF(2,24)=13.45, p<0.001). Further analyses revealed that both 1AKO and1BKO mice were faster in acquiring the autoshaping procedure. Inaddition, the mean number of nose pokes per minute, a putative measureof activity, was different between genotypes (ANOVA, F(2,24)=12.14,p<0.001), with 1BKO making the most nose pokes per minute (FIG. 9B). Anon-parametric correlation (Kendall's τ) indicated that nose pokebehavior and time needed to reach criterion were negatively correlatedin all genotypes (r=−0.88, p<0.001). The increased nose poke respondingin 1BKO mice reflects an autoshaped conditioned response and may be aform of impulsive responding (Tomie et al., Psychopharmacology,139:376-382, 1998). Nose poke activity was not significantly increasedin 1AKO mice during autoshaping phase.

FIG. 10 shows the difficulty the 1BKO mice have, in contrast towild-type or 1AKO, learning the DL-36s schedule. The 1BKO mice exhibitedsevere time discrimination problems. The 1BKO mice consistently had ahigher response rate, as depicted in FIG. 10A, and a lower reinforcementrate, as depicted in FIG. 10B, over the course of the first fourteensession compared to wild-type or 1AKO mice, indicating poor acquisitionof the task. By contrast, wild-type and 1AKO mice progressivelyresponded less frequently, as they learned the DRL 36s, waiting theappropriate interval in order to receive reinforcement. As aconsequence, the wild-type and 1AKO mice received more reinforcementsover the course of training. FIG. 10A shows that response ratesdecreased in all genotypes (F(6,114)=15.70, p<0.001), however theblock×genotype interaction effect was not significant (F(12,114)=1.50,p=0.14). Moreover, there was a significant difference in response ratesbetween genotypes (F(2,19)=20.15, p<0.001). Post hoc comparisonsindicated that all genotypes differed, with 1BKO mice having the highestresponse rates and WT having the lowest response rates. The number ofreinforcements were significantly increased for wild-type and 1AKO, butnot for 1BKO mice (F(6,114)=16.48, p<0.001; block×genotype interactioneffect, F(12,114)=4.06, p<0.001). Response rate of all genotypesdiffered significantly from one another (F(2,19)=22.06, p<0.001).

FIG. 10C shows that 1BKO mice did not lengthen their wait time torespond until towards the end of training, unlike the wild-type and 1AKOmice. Peak deviation analysis showed that PkL increased over blocks inall mice (F(6,114)=2.73, p<0.05), but there was a significant genotypedifference (F(2,19)=17.12, p<0.001); there was no significantblock×genotype interaction effect (F(12,114)=1.46, p=0.15).

Once the DRL performance had stabilized, the response rate of 1BKO miceremained significantly higher and the reinforcement rate lower thanwild-type or 1AKO mice. The IRT histograms of stable DRL performance ofwild-type, 1BKO, and 1AKO mice are shown in FIGS. 11A, 11B, and 11C,respectively. FIG. 11 demonstrates that the PkL remained much shorter(22.4 sec) than wild-type (35.2 sec) or 1AKO mice (34.0 sec). Insummary, a mouse (like the 1BKO) showing a disruptive DRL-strategy is auseful model for assessing the ability of a drug to alter animalbehavior which may reflect the hyperactivity-impulsivity symptoms ofADHD. Thus, the DRL-36s schedule can be used to measure anti-impulsiveeffects of psychotropic agents on 1BKO mice. The behavior of 1BKO micein the DRL-36s paradigm is in general agreement with the type ofbehaviors observed in ADHD children, indicating 1BKO mice are a usefulanimal model of ADHD.

EXAMPLE 8

Since impulsivity with hyperactivity are behaviors associated with ADHD,1BKO mice were further tested on a DRL-36s schedule to evaluate thepotential therapeutic effect of eltoprazine. The effects of eltoprazineand d-amphetamine on the performance of 1BKO and wild-type mice onDRL-36s schedule were compared to determine whether these drugsinfluenced the behavior of 1BKO and wild-type mice in the same manner.

Behavioral Testing

Mice were conditioned and challenged on a DRL-36s schedule according tothe methods of Example 7, with the following modifications. Because 1BKOmice had difficulty learning the DRL-36s schedule following conditioningon an FR1, wild-type and 1BKO mice were conditioned on a fixed rate 5(FR5) schedule. In FR5, instead of pressing the lever once for a reward,animals have to press 5 times. The rationale for changing the operantconditioning was that by increasing the number of responses necessaryfor reinforcement, the wild-type mice should have greater difficulty andthe 1BKO (which have higher response rates anyway) should have lessdifficulty getting reinforcements. In fact, we found that following FR5conditioning, wild-type and 1BKO mice did not differ dramatically intheir DRL-36s performance.

Results

d-Amphetamine, at 2 and 8 mg/kg, significantly decreased the number ofreinforcements in wild-type mice but had no significant effect on numberof reinforcements in 1BKO mice, as depicted in FIG. 12. ANOVA revealed asignificant dose main effect (F(3,92)=3.53, p<0.05) and a significantdose×genotype interaction (F(3,92)=4.44, p<0.01), but genotype maineffect was not significant. The effect of amphetamine on response ratesin 1BKO mice was not statistically significant at any dose, butamphetamine significantly increased response rate in wild-types at 2mg/kg and decreased response rate in wild-types at 8 mg/kg. At 4 mg/kg,amphetamine significantly decreased response rate in wild-types comparedto 1BKO mice. ANOVA revealed significant dose main effect (F(3,92=17.0,p<0.001), dose×genotype interaction (F(3,92)=3.48, p<0.05), and genotypemain effect (F(1,92)=5.26, p<0.05). The increased response rate anddecreased reinforcement rate at the lowest dose of amphetamine isindicative of disruption of the behavior.

By contrast, eltoprazine had comparable effects on the behavior ofwild-type and 1BKO mice in the DRL-36s task, as depicted in FIG. 13.Eltoprazine significantly decreased reinforcements equivalently inwild-type and 1BKO mice at 0.5 mg/kg and 1 mg/kg, and significantlydecreased response rates equivalently in wild-type and 1BKO mice at 0.5mg/kg in the DRL-36s schedule. ANOVA revealed a significant dose maineffect for reinforcements (F(3,57)=8.57, p<0.001) and response rates(F(3,92)=5.59, p<0.001), but no significant genotype main effect ordose×genotype interaction for either reinforcements or response rates. Acomparison of FIGS. 12 and 13 demonstrate that eltoprazine exhibitsamphetamine-like properties in both genotypes.

To summarize, the wild-type and 1AKO mice learn to wait at least 36seconds before responding and receive a greater number ofreinforcements. By contrast, the 1BKO knockout mice respond too earlyand their behavior does not improve very much over successive trials,indicative of their hyperactive-impulsive tendency and the lack ofinvolvement of 5-HT_(1B) receptors. Eltoprazine shares some of theproperties of d-amphetamine in its effects on DRL-36s behavior.Similarities are particularly notable in the number of reinforcements,where genotype-independent decreases are present. The eltoprazineprofile is different from d-Amphetamine, however, perhaps reflecting ananti-ADHD profile distinct from psychostimulants.

EXAMPLE 9

To further determine what if any role 5-HT_(1B) receptors play in theanti-ADHD effects of eltoprazine of this invention, in the context of DAand 5-HT interactions, DA and 5-HT release were examined in eltoprazinetreated mice using in vivo microdialysis. Eltoprazine-induced changes inbasal DA and 5-HT release were then compared with the effects of aspecific 5-HT_(1B) receptor agonist CP 93129. In vivo microdialysispermits measurement of extracellular neurotransmitter concentrations inawake, freely moving animals.

In Vivo Microdialysis and HPLC-ECD Analysis

Wild-type and 5-HT_(1B) knock out mice were implanted with microdialysisprobes according to the methods of DeGroote et al., incorporated hereinby reference, with the following modifications. (DeGroote et al., Eur.J. Pharmacol., 439:93-100, 2002). The dialysis probe was placed in thedorsal striatum, at coordinates AP +0.80, ML −1.7 mm from bregma, DV−4.0 mm from the dura, according to the stereotaxic atlas of the mousebrain (Franklin and Paxinos, 1997), and with the toothbar set at 0 mm.Microdialysis experiments were begun 16-20 hours after surgery, usingpreviously described methods, which are incorporated herein by reference(DeGroote et al., 2002). After the start of the dialysis probeperfusion, mice were left undisturbed for three hours. Mice were testedin their home cage during the light period. Samples were collected every20 minutes in vials containing 7.5 μl acetic acid and stored at −80° C.until HPLC analysis.

Release of DA and 5-HT was measured after peripheral administration ofeltoprazine or intrastriatal administration of the selective 5-HT_(1B)receptor agonist CP 93129 dihydrochloride(1,4-Dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo[3,2-b]pyridin-5-one,obtained from Tocris, UK). Drugs were dissolved in distilled water andfurther diluted in Ringer solution to the final concentration on the dayof the experiment. 5-HT and DA were analyzed by HPLC withelectrochemical detection. Samples (25 μl) were injected onto anInertsil ODS-3 column (3 μM, 2.1×100 mm, Aurora Borealis, TheNetherlands) using a Gilson pump and autosampler (Separations, TheNetherlands). Separation was performed at 40° C. with theelectrochemical detector (Intro, ANTEC Leyden, The Netherlands) set at apotential of 600 mV against an Ag/AgCl reference electrode. The signalwas analyzed using Gynkotek software. The mobile phase consisted of 5g/l (NH₄)₂SO₄, 150 mg/l heptane sulphonic acid sodium salt, 0.5 g/lEDTA, 5% methanol, 30 μl/l triethylamine, 30 μl/l acetic acid, pH 4.6.Flow rate was 0.3 ml/min. The detection limit for 5-HT was 0.5 fmol/25μl sample (signal to noise ratio 2).

Values for the first four consecutive microdialysis samples wereaveraged to calculate the basal levels of extracellular 5-HT and DA,uncorrected for probe recovery. Student's t-tests were used to comparebasal 5-HT and DA values between the two genotypes. Effects of drugtreatment were analyzed by a repeated multivariate analysis of variance(ANOVA) with time as ‘within’ and treatment (or dose) and genotype as‘between’ factors.

Results

Basal levels of extracellular 5-HT and DA levels in the dorsal striatumwere not different between wild-type and 1BKO mutants. Basal 5-HT levelswere 4.0±0.3 fmol/sample in wild-type and 5.0±0.6 fmol/sample in 1BKOmice. Basal DA levels were 181.3±14.6 fmol/sample in wild-type and183.1±15.9 fmol/sample in 1BKO mice.

Eltoprazine effects on DA and 5-HT release were similar and independentof genotype. Eltoprazine (0.1 mg/kg, i.p.) decreased basal release ofboth DA and 5-HT release in dorsal striatum of awake wild-type micewithin 20 minutes after administration, as depicted in FIG. 13. In theseanimals, striatal release of DA and 5-HT remained below basal levels forat least 100 minutes after drug administration.

In contrast to eltoprazine, CP 93129 had differing effects on 5-HT andDA release that were genotype specific at low dose. Local administrationof CP 93129 (0.5 μm) decreased striatal 5-HT release in wild-type mice(FIG. 14), an effect similar to that observed with eltoprazine. Repeatedmeasures ANOVA revealed a dose main effect (F(1,17)=6.2, p<0.05) andgenotype main effect (F(1,17)=13.5, p<0.01). In wild-type mice, CP93129(0.5 μM) reduced 5-HT to 51±9% as compared to vehicle (F(1,10)=11.2,p<0.01). 5-HT release returned toward basal levels within 40 minutesafter cessation of CP 93129. In contrast to eltoprazine, however, DArelease was unaffected by 0.5 μM CP 93129 (FIG. 15). The CP 93129attenuation of basal 5-HT release was mediated through the 5-HT_(1B)receptor. As FIG. 14 illustrates CP 93129 did not decrease 5-HT releasein mice lacking the 5-HT_(1B) receptor (i.e., 1BKO).

Striatal release of DA in 1BKO mice was similarly unaffected by CP 93129(FIG. 15). As FIG. 15 further shows, a very high concentration of CP93129 (50 μM) increased DA release in striatum of wild-type mice. ThisCP 93129-induced stimulation of DA release was independent of 5-HT_(1B)receptor activation, since a comparable increase in DA release wasobserved in 1BKO mice. A repeated measures ANOVA of the CP 93129 datarevealed dose main effect for (F(2,32)=55.6, p<0.001) and a time×doseinteraction effect (F(16, 256)=7.9, p<0.001), but not a genotype maineffect (F(2,32)=0.5, p=0.98). The high dose of CP 93129 increased DAlevels in 1BKO mice to the same extent as in wild-types when compared tovehicle (p<0.001). These CP 93129-induced increases were 525±79% and527±67% in wild-type and 1BKO, respectively.

In summary, because eltoprazine affects both 5-HT and DA release, it ispresumed that its mechanism of action in alleviating behaviorsassociated with ADHD is different from its 5-HT_(1B) agonist properties.Moreover, while both eltoprazine and the psychostimulant anti-ADHD agentamphetamine attenuate ADHD-associated behaviors, inattentiveness, andhyperactivity with impulsivity, the mechanism of action of eltoprazinemay be differentiated from that of amphetamine. Eltoprazine was observedto decrease striatal DA release, whereas amphetamine is well known toincrease extracellular striatal DA levels (see Hess et al., 1996,supra).

The above Examples are for illustrative purposes only and are notintended to limit the scope of the invention.

1. A method of treating Attention-Deficit/Hyperactivity Disorder(“ADHD”) in humans by administering a therapeutically effective amountof a compound according to the formula

wherein R₁ is hydrogen, alkyl, cycloalkyl, optionally esterifiedhydroxyalkyl, alkoxyalkyl, optionally substituted phenyl or heteroaryl,alkoxy, aryloxy, alkylthio, arylthio, acyl, alkoxycarbonyl,aminocarbonyl, alkyl- or dialkyl-aminocarbonyl, nitro, amino, alkyl- ordialkyl-amino, acylamino, alkylsulfonylamino, arylamino, cyano, halogen,trifluoromethyl, trifluoromethoxy, optionally esterified hydroxyl,alkyl- or amino-sulphonyl or -sulphinyl, alkyl- ordialkyl-aminosulphonyl or -sulphinyl, and p has the value 0-3; R₂ andR′₂ are independently hydrogen or an alkyl group and n and q can havethe value 0 or 1; R₃ may have the same meaning as R₁, or is alkylidene,an oxo or thioxogroup, and m has the value 0-2; A forms, with the twocarbon atoms of the phenyl group, an optionally entirely or partlyunsaturated cyclic group having 5-7 atoms in the ring, which comprises1-3 hetero atoms from the group O, S, and N, with the proviso that thesum of the number of oxygen and sulphur atoms is at most 2; wherein thecompound may be a racemate or a single diastereomer or enantiomer; or apharmaceutically acceptable acid addition salt thereof. 2-18. (canceled)